Isoelectric focusing ("IEF") also sometimes called electrofocusing, is an electrophoretic technique that is recognized as being a powerful method for the analysis and micropreparative separation and purification of various biological materials, including proteins, peptides, nucleic acids, viruses, and even some living cells of cell organelles. The principle of IEF is based on the fact that certain biomaterials, such as those listed above, are amphoteric in nature, i.e. are positively charged in acidic media and negatively charged in basic media. At a particular pH value, called the isoelectric point, they have a zero net charge. In other words, the isoelectric point is the pH value at which they undergo a reversal of net charge polarity. In a pH gradient such materials will migrate under the influence of a d.c. electric field until they reach the pH of their isoelectric point where they become immobilized by virtue of their zero net charge. Thus, they focus into narrow zones, defined by the pH of the medium and the electric field applied.
IEF techniques have been greatly advanced by the development of suitable buffer systems which form stable pH gradients in the electric field. Such buffers are usually composed of a random mixture of amphoteric substances having isoelectric points covering a wide spectrum of pH values. In the electric field, these components of the buffer mixture are also focused according to their isoelectric points, thereby establishing a stable pH gradient. A commercial mixture of such amphoteric substances called "Ampholine" is available from LKB Produkter AB, a Swedish Company. Other buffer systems are also compatible with IEF. The electric field in IEF thus has two simultaneous and overlapping functions; these being the establishment of the pH gradient and the focusing of the biomaterials to be separated. In terms of time sequence, the establishment of final focusing of the biomaterials cannot be achieved before a stable pH gradient is formed, i.e. before the components of the buffer mixture are focused.
IEF separation processes may be disturbed by bulk flow of liquid within an IEF apparatus. Two potential causes of bulk flow are electroosmosis and convection. If unchecked, these can disrupt the separation process by causing remixing of the separated fractions. Electroosmosis is caused by an electrical charge on the walls of the separation vessel, while convection is caused by local density differences arising from temperature and/or solute concentration gradients.
To prevent these disturbances, analytical IEF is mostly carried out in gels or packed granular support media. For preparative applications, free solutions are often preferred, as they simplify the collection of separated fractions. To control disruptive effects in free solution, several approaches have been previously used, whether for IEF or for more conventional forms of electrophoresis.
In vertical columns, density gradients have been very effective for fluid stabilization. These are formed by stratifying solutions of an electrically neutral solute, in order of decreasing density. While this system is quite effective for IEF, collection of the separated fractions is slow and cumbersome, a rapid drainage of the column frequently causing partial remixing of the separated fractions.
Shear effects have been utilized for fluid stabilization. The fluid is confined to a narrow space between two parallel plates as, for instance, in U.S. Pat. No. 4,310,408 to Rose, et al. This type of electrophoretic instrument has been utilized only rarely for IEF.
In horizontal columns, rotation around an horizontal axis has minimized convective disturbances by continuously changing the relationship of the fluid to the gravity vector. This mode of stabilization protects only against convective flows but not against electroosmosis. To minimize the same, anticonvective agents such as agarose or dextran are introduced either in the medium or as coatings on the column walls. Mainly, this mode of fluid stabilization is effective only in small bore tubes, as first shown by Hjerten (S. Hjerten: "Free Zone Electrophoresis", Almqvist and Wiksells, Uppsala, 1967) and collection of fractions is quite difficult (U.S. Pat. No. 4,040,940 to Bier).
A recirculating mode of focusing has been recently described in my previous U.S. Pat. Nos. 4,204,929 and 4,362,612 the disclosures of which are incorporated herein by reference. The solution to be fractionated is recirculated through a multichannel focusing cell and heat-exchange reservoir, by means of a multichannel peristaltic pump. While quite effective, relatively large volumes of fluid are necessary to prime the apparatus. In many research applications, where only small quantities of protein are available, this forces excessive dilution of the solution. Dilute protein solutions are notoriously less stable than more concentrated solutions. Experiments have shown that some recirculation or other form of mixing of the contents of each subcompartment is desirable, as separation in static fluids produces inferior results. Using colored proteins, such as hemoglobin, one notices a `gravitational slumping` of the focused protein across the separating filter elements, if recirculation is interrupted.
Accordingly, an object of the present invention is to provide a novel method and means for stabilizing IEF columns against both electroosmosis and convection, which is applicable to both small and large processing volumes.
Another object of the invention is to provide means for preparative isoelectric focusing in free fluids, without the need for supporting materials, such as gels, powders, etc.
A further object of the invention is to provide means for rapid focusing of small volumes of proteinaceous liquid.
A still further object of the invention is to provide a means for simple and rapid collection of separated fractions.